Multi-target molecular mechanisms of Cymbopogon flexuosus essential oil reveal membrane destabilization, ROS-mediated cellular damage, efflux perturbation, biofilm suppression and resistance-target interference in Staphylococcus aureus.

The rapid emergence of antibiotic-resistant Staphylococcus aureus necessitates exploring alternative antimicrobial agents with multi-target mechanisms of action. In the present study, the antibacterial efficacy and mechanistic activity of 21 essential oils were investigated against S. aureus using integrated microbiological, biochemical, flow cytometric, microscopic, and computational approaches. Preliminary screening through disc diffusion, MIC, and MBC assays identified Cymbopogon flexuosus, Cymbopogon nardus, and Eucalyptus hybrida as the most active essential oils, with C. flexuosus exhibiting the strongest antibacterial potency. Percentage inhibition assays, growth kinetics, confirmed concentration-dependent antibacterial activity, with C. flexuosus and C. nardus producing > 60% inhibition and rapid growth suppression within 2 h at MIC and 2×MIC. Mechanistic investigations revealed that C. flexuosus essential oil induced significant membrane destabilization, as evidenced by the crystal violet permeability assay, as they increase relative permeability 58.2% at twice MIC, increased nucleic acid and protein leakage, increased propidium iodide permeability, and elevated ethidium bromide accumulation, indicating membrane disruption and perturbation of efflux pumps. Scanning electron microscopy further confirmed severe morphological alterations, including membrane collapse and cellular deformation. In addition, intracellular reactive oxygen species (ROS) generation increased significantly following treatment, suggesting oxidative stress-mediated bacterial killing. C. flexuosus also demonstrated potent antibiofilm activity, reducing biofilm formation by more than 60% at the MIC and twice the MIC in S. aureus. GC-MS analysis identified geranial, neral, citronellal, geraniol, limonene, and β-caryophyllene as major volatile constituents. Molecular docking studies revealed favourable interactions between several compounds and critical bacterial targets involved in cell wall biosynthesis and antibiotic resistance, including β-lactamases and penicillin-binding proteins. Furthermore, in silico ADMET profiling suggested that the selected constituents possess favourable physicochemical and safety properties for topical therapeutic applications. These findings demonstrate that C. flexuosus essential oil exerts potent anti-staphylococcal activity through multiple mechanisms, including membrane destabilisation, ROS-mediated cellular damage, efflux perturbation, biofilm suppression, and interference with resistance targets. The study highlights the therapeutic potential of C. flexuosus essential oil as a promising natural antibacterial candidate against S. aureus infections.